Paramyxoviruses cause important human illnesses that contribute significantly to global disease and mortality. Two zoonotic paramyxoviruses, Hendra (HeV) and Nipah (NiV), are of urgent concern due to their lethal and transmissible nature. HeV and NiV initiate infection by binding to cell surface receptors, and fuse directly with the cell membrane to enter. The receptor-binding molecule (G) triggers the viral fusion protein (F) to its active state, and conformational changes in F protein drive fusion. Molecular mimics of the heptad repeat (HR) regions of HeV F can prevent F from reaching fusion-ready conformation, and prevent infection. We found that a heterologous (parainfluenza 3) peptide is more effective than the homologous peptide as an anti-HeV/NiV. We propose a distinctive combination of: (1) experimental and structural analysis of the molecular mechanisms of fusion and entry inhibition, to design optimal inhibitors; (2) animal model studies to test the proposed antivirals for protection from infection; (3) bioengineering approaches to improve delivery systems for promising antivirals. A multidisciplinary collaborative team, bringing unique expertise, synergizes to study: 1. Conformational changes in HeV and NiV F-protein: Basic research to design effective peptides. 1.1 Structural analysis of the mechanism of fusion inhibition by homotypic and heterotypic HRC peptides. By combining experimental information with analysis of our X-ray crystal structures of the 6HBs of HPIV3 F, HeV F and HPIV3/HeV chimeric 6HBs, the mechanism of action of inhibitory peptides will be explored structurally and biophysically. 1.2 Design and testing of improved peptides based on crystal structure data. 1.3 Analysis of HPIV3 HRC fusion inhibition-resistant variants. 2. Effectiveness of the peptides to protect from live viral infection in vivo. Effective peptides (aim 1) will be tested for their ability to protect against infection with HeV and NiV infection in the ferret and cat models of acute HeV/NiV infection. Treatment as well as pre- and post-exposure prophylaxis will be addressed. Micro/nanoparticles will be engineered to provide sustained delivery of the most promising inhibitors, to explore the hypothesis that sustained release can improve antiviral efficacy in vivo and provide a clinical strategy that would be feasible for crisis situations. Feedback from aim 2 will lead directly to new experiments in aim 1. The results will lead to: (1) New understanding about the molecular mechanisms of virus fusion, entry, and mechanisms of action of peptide inhibitors; (2) sustained delivery systems for antivirals that may be broadly applicable and clinically relevant; (3) validation of an antiviral strategy in vivo, and identification of realistic candidate antiviral peptides. The results will be significant in light of the importance of paramyxoviruses to human health and the potential broad applicability of the new platforms, in addition to the specific clinical/biodefense relevance of these emerging zoonotic pathogens.